PROJECT SUMMARY - PROJECT 2 The initial interaction of JCPyV with its host involves recognition of specific receptor complexes on cells. Recognition of these receptors leads to virus penetration of the host cell membrane, trafficking of the virion to the endoplasmic reticulum, and the eventual delivery of the dsDNA genome to the cell nucleus. Our team, supported by this program project, elucidated the critical components of the JCPyV receptor complex and determined that the major capsid protein VP1 was responsible for directing the virus to the ER. We developed novel tools to study not only lab adapted strains of JCPyV but also mutant forms of the virus that have been reported to arise in the brains of patients with PML. These mutant forms of JCPyV have lost the ability to recognize the sialic acid receptor and as a consequence are no longer infectious in most cell types examined. We hypothesize that these mutants either recognize alternative receptors which are yet to be identified or that they have gained the capacity to spread directly from cell to cell bypassing the requirement for cell surface receptors. Our approach in project 2 is to use pseudoviruses developed in core B to further explore potential receptor usage by these mutants. We will also explore the possibility that these mutants, are capable of direct cell to cell spread by engineering the mutations into an infectious JCPyV clone and following their growth after transfection of the genomes into different cell types grown as confluent monolayers. Regardless of the mechanism of infection (receptor mediated OR direct cell to cell spread) these viruses must all traffic to the ER to begin the uncoating process for eventual delivery of their genomes to the nucleus. In the last funding cycle we discovered that the dihydroquinozolinone compound Retro-2cycl potently prevents JCPyV trafficking to the ER and substantially reduces initial infection and infectious spread. Our goal now is to define its mechanism of action, to identify its cellular targets, and to optimize the existing compound so that a therapeutic window of inhibition can be determined.